To confirm VGLUT1 specificity for afferent synaptic inputs we analyzed VGLUT1-immunoreactivity around calbindin-IR Renshaw cells in P15 Er81-/- mutants that lack ventral horn sensory afferent projections (Arber et al., 2000) and compared them to wild-type age-matched littermates. Wild-type (Er81+/+) and mutant (Er81-/-) spinal cords did not differ in overall morphology, density or distribution of NeuN-immunoreactive neurons (we used a mouse monoclonal antibody, Chemicon, dilution 1:1000) or calbindin-IR Renshaw cells (panels A-D; Er81+/+: 5.9 ± 0.3 Renshaw cell profiles per ventral horn in 40 µm thick sections compared to 5.1 ± 0.7 in Er81-/- mutants; p>0.05, t-test). VGLUT1-immunolabeling in Er81-/- mutants was restricted to the dorsal horn (panel B). Correspondingly, VGLUT1-IR contacts on Renshaw cells dropped to almost zero in Er81-/- mutants (panels E-G). In addition to proprioceptive primary afferents, corticospinal tract (CST) neurons also express VGLUT1 and Er81 (Fremeau et al., 2001; Yoneshima et al., 2006). The lumbar ventral horn receives a minor CST projection after P6 (Bareyre et al., 2005). The stability of CST projections in Er81-/- mutants is unknown, but this sparse projection is clearly outnumbered by the high density of ventral VGLUT1-IR boutons. Moreover, most VGLUT1-IR varicosities in lumbar 5 and 4 ventral horns distribute within lateral laminae IX and VII, while the ventral CST projects to medial laminae VII and VIII. Taken together, the data suggest that VGLUT1-IR contacts on Renshaw cells originate from Er81-dependent (proprioceptive) primary sensory afferents. Figure legend : A, B, Low magnification images of P15 spinal cords from wild type (Er81+/+, A) and mutant (Er81-/-, B). Neuronal cell bodies were immunostained with antibodies against NeuN (Cy3, red) and superimposed on VGLUT1-IR boutons (FITC, green). No major differences in distribution or densities of spinal neurons were observed, however, VGLUT1-IR boutons were reduced in Er81-/- ventral horns. C, D, Medium magnification images showing calbindin-IR Renshaw cells (red) and VGLUT1-IR boutons (green, VF, ventral funiculus). Renshaw cell numbers, distribution and calbindin-immunoreactivities showed no differences. VGLUT1-IR boutons were depleted from the Renshaw cell area. E, High magnification images of calbindin-IR Renshaw cells (red) receiving contacts from VGLUT1-IR boutons (green) in the wild-type. F, Almost no contacts were observed in Er81-/-. The rare contacts identified on the dendrites of Renshaw cells from Er81-/- knockout mice were rather small (arrow in F). G, VGLUT1-IR contact densities were dramatically reduced on both dendrites and somata in Er81-/- knockout animals (t-test; p<0,001, n=10 Renshaw cells in wild type and in Er81-/- knockout; error bars indicate SEM). Scale bars, 200 µm in A (B at same magnification); 100 µm in C,D; 20 µm in E,F. References: Arber S, Ladle DR, Lin JH, Frank E, Jessell TM (2000) ETS gene Er81 controls the formation of functional connections between group Ia sensory afferents and motor neurons. Cell 101: Bareyre FM, Kerschensteiner M, Misgeld T, Sanes JR (2005) Transgenic labeling of the corticospinal tract for monitoring axonal responses to spinal cord injury. Nat Med 11: Fremeau RT, Jr., Troyer MD, Pahner I, Nygaard GO, Tran CH, Reimer RJ, Bellocchio EE, Fortin D, Storm-Mathisen J, Edwards RH (2001) The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron 31: Yoneshima H, Yamasaki S, Voelker CC, Molnar Z, Christophe E, Audinat E, Takemoto M, Nishiwaki M, Tsuji S, Fujita I, Yamamoto N (2006) Er81 is expressed in a subpopulation of layer 5 neurons in neocortex. Neuroscience 137: